Chronic pain is estimated to cost the U.S. economy approximately $635 billion per year, with over $300 billion of this being direct costs for medical care. This surpasses the total US expenditure for heart disease, cancer, and diabetes combined. Pain is the most prevalent symptom presented at hospitals, and over 1.5 billion people in the world, and 116 million people in the United States, suffer from chronic pain. All people are at risk of chronic pain, and patients suffering from chronic pain experience a significant reduction in their quality of life. Additionally, pain is comorbid with the diseases mentioned above and many others, such as gastrointestinal disease, arthritis, and neuropsychiatric disorders, complicating the management of these diseases.
Pain has been managed primarily by opioid drugs for over two centuries, but their use is highly limited by adverse effects. Morphine, isolated in 1806, is still the mainstay of our management of pain. Morphine activates the mu-opioid receptor (MOR) in the nervous system. Over the last hundred years, we have generated over a thousand different analogs that activate the same target receptor, which potentially can act as effective analgesics for acute pain relief. In practice, however, the use of these drugs has been highly complicated by the rapid development of adverse effects. These include symptoms such as nausea, constipation, and dizziness, as well as the serious side effect of the development of addiction to opioid drugs. Patients develop tolerance (i.e., they need higher and higher doses to get the same analgesic effect) and dependence (i.e., they get adverse “withdrawal” symptoms in the absence of drug) to these drugs. There is no real strategy to treat addiction, and it is a major socioeconomic problem in the world today.
Addiction to opioids is a highly prevalent and comorbid disease that adds an additional cost to pain management. An estimated 10-15 million people in the US are addicted to prescription opioids, causing an additional revenue loss of $193 billion per year, with about $11 billion in healthcare costs. This is a rising trend for the future, with 26% of adolescents in schools reporting the use of illicit drugs in 2013. Overall, 52 million (i.e., 1 in 6) adults in the US have used prescription opioids without pain symptoms or a prescription, and 1 in 15 of these is expected to try heroin in the next 10 years. Consistent with this trend, opioid overdose deaths have shown almost a 100% increase over the last 10 years, up to 16,000 in 2010. Currently, the only method of managing addiction is to maintain the patient on methadone, often considered a less harmful alternative. This is far from ideal, and is further complicated by lack of patient compliance and an increased risk of abuse and deaths from methadone in the recent years.
The analgesic and addictive effects of MOR activation have not been separated, despite many years of efforts following different hypotheses. Addiction arises from the fact that MOR also activates the reward pathway in the brain, causing dependence of the patient to opioids. Peripherally restricted opioids have been tried to counter this, but they are only partially effective, and also initiate severe adverse effects. A second aspect of addiction is tolerance, where increasing doses are required to generate the original efficacy. Development of dependence and tolerance severely limit the long-term use of opioids in chronic pain. Importantly, the thousands of opioid analogs that have been generated, as part of a decades-long drive to find a non-addictive analgesic, all share the same limitations. Therefore, the identification of an alternate target for managing pain will have an immediate and profound socioeconomic impact.
The delta-Opioid Receptor (DOR) has been long-considered a promising alternate target for pain relief. DOR agonists are relatively non-addictive, and have much fewer adverse effects, because they are thought to not activate the reward pathway. The exact contribution of DOR-expressing neurons towards different modalities of antinociception is still being explored, but it is clear that these neurons mediate at least the main modalities such as mechanical pain. At a molecular level, DOR acts through the same pathways as MOR, reducing cAMP (cyclic adenosine monophosphate) and inhibiting Ca2+ channels, and opening K+ channels, to hyperpolarize and inhibit neurons. Although DOR agonists have relatively similar efficacies as MOR agonists in isolated systems, administrations of either peripheral (e.g., DADLE, loperamide) or central (e.g., SNC-80) agonists have poor analgesic responses in vivo. Further, at high concentrations, centrally acting DOR agonists can minimally inhibit pain, but also induce adverse side effects like convulsions, though a few DOR agonists have recently been developed that do not show such unwanted side effects. But the lack of effectiveness in vivo remains the limiting factor in targeting DOR for pain management.
Additionally, DOR dysfunction might also play roles in depression and related neuropsychiatric disorders. But drugs targeting this receptor have not been effective in vivo. There are four aspects to the current understanding of DOR:
1) Published data (since about the mid-2000's) indicate that most of the DOR in a neuron is kept in intracellular “storage” pools, and is not present on the surface. DOR is retained in intracellular locations in neurons in the periaqueductal gray and the dorsal horn of the spinal cord, areas that are important for pain sensation, with limited expression on the surface. As DOR needs to be on the cell surface to bind drugs and activate surface-localized effectors (e.g., adenylate cyclase) and analgesic pathways, this lack of surface localization could be a major contributor to the low effectiveness of DOR agonists, preventing efficient analgesia in vivo.
2) DOR agonists that can cross the blood brain barrier cause adverse reactions like convulsions at doses that are effective as analgesics. DOR agonists that cannot cross the blood brain barrier (i.e., peripherally restricted agonists) have not been effective, but it is not clear whether this is due to low availability of DOR in peripheral neurons.
3) In conditions of chronic pain, there is an increase in total expression of DOR, which leads to a proportionally higher amount being available on the surface, and therefore a proportional increase in efficiency for DOR agonists. Consistent with this, there is a correlation in an increased DOR expression on the surface, induced by pathological conditions such as inflammation or substance abuse, to better analgesic/antinociceptive effects of DOR agonists. Typically, such scenarios (e.g. inflammation), are thought to “prime” neurons to DOR agonists by increasing total DOR expression, and, therefore, proportionally increasing the amount on the neuronal surface, allowing DOR agonists to reduce hyperalgesia and relieve allodynia. Even in these cases, the effect is still less than clinically useful efficiencies. However, this leads to the hypothesis that, if one can find strategies to force DOR delivery to the neuronal surface, it will be an efficient alternate target for managing pain.
Importantly, while these speak to the feasibility of increasing DOR on the surface to target pain, there has been no previous attempt to change the trafficking of DOR to increase the proportion of surface DOR under normal situations. This is largely because the mechanisms regulating DOR trafficking and surface delivery have been poorly understood.
In addition, it is known that physiological responses of cells to changes in the extracellular environment rely on the precise localization of transmembrane receptors. Due to the amount of energy required for synthesis and trafficking of new proteins from the endoplasmic reticulum to the surface, one might intuit that the cell would re-use these receptors multiple times. While many receptors are recycled and re-used, however, this is not always the case. Many receptors are delivered to the cell surface and activated just once, before being destroyed. Whether a receptor is “multi-use” or “single-use” is determined by specific protein sequences on the receptors that result from small genetic variations between otherwise similar proteins.
Relevant examples of proteins with similar function, but different trafficking characteristics are the mu- and delta-Opioid Receptors (MOR and DOR), each part of the Gi-coupled G Protein-Coupled Receptor (GPCRs) subtype. In each case, the activated Ga subunit decreases the activation of adenylyl cyclase and cyclic-AMP production inhibiting signaling pathways within the cell. Additionally, activation of the opioid receptors inhibits calcium channel influx and promotes potassium channel efflux resulting in neuronal hyperpolarization that inhibits action potential initiation.
Opioid receptors are typically activated in our nervous system by endogenous endorphin or enkephalin agonists or by an exogenous opioid. While both opioid receptors can mediate pain inhibition, the mu-Opioid Receptor (MOR) has been more commonly targeted for drug development, and MOR agonists have been far more effective. Upon activation by an exogenous opiate agonist, MOR signaling is activated, and receptors are removed from the cell surface via endocytosis and transferred to intracellular endosomes. This leads to the loss of sensitivity of neurons to MOR agonists, because the surface receptor number is reduced. From the endosome, however, MOR is recycled to the cell surface for another round of agonist binding and signaling. Therefore, this recycling is key for recovery of the sensitivity of neurons to signaling by drugs that target MOR. In contrast, DOR's trafficking differs from MOR's trafficing in that after activation and endocytosis into the early endosome, it is excluded from recycling and instead is targeted to the lysosome where it is degraded. Therefore, DOR is a prototypical “single-use” receptor. Importantly, in order to obtain more DOR on the cell surface, newly synthesized receptors must be synthesized, trafficked through the biosynthetic pathway, and delivered to the cell surface.
Because the biosynthetic pathway regulates the sensitivity of neurons to DOR, unlike MOR, the delivery of the DOR from intracellular stores, as mentioned above, is critical. This difference in trafficking between DOR and MOR might be clinically relevant in chronic pain therapy, because the increased MOR desensitization and tolerance seen with habitual opiate administration is associated with changes in MOR endocytic trafficking and recycling. Thus, due to DOR's single-use characteristics, targeting of DOR might, among other treatments, provide a method to circumvent the potential addictive tendencies of opiates arising from MOR desensitization. The critical limitation is that the bioavailability of DOR on the surface of neurons, where they can be activated by drugs, is limiting, because DOR is stored in intracellular pools in neurons. Therefore, a method whereby the retained pool of intracellular DOR can be released and transported to the cell surface is desirable.
As such, a need exists to increase the bioavailability of DOR, and, in particular, to inducing the surface trafficking of DOR in neurons. The present invention addresses this need by (1) identifying drug-targetable enzymes that contribute to DOR retention and reduced surface expression, and (2) identifying a method to drive DOR to the surface of neurons. As a result, DOR agonists can be used at lower effective doses as peripheral or systemic analgesics. The DOR is also made more readily available for treatment of other disorders as well.